Project Summary/Abstract The electron transport chain (ETC) activity in mammalian cells is necessary for survival and proliferation. The ETC is composed of ~100 subunits mostly encoded in the nuclear genome, but 13 essential subunits are in the mitochondrial genome (mtDNA). Accumulation of mutations in the mtDNA can lead to severe genetic defects and cell death. Interestingly, we and other groups have found the occurrence of loss-of- function (LOF) mtDNA mutations across a variety of cancer types at high heteroplasmy. Heteroplasmy is defined as the proportion of mtDNA with a specific mutation over the total number of mtDNA. Furthermore, there is an enrichment for these LOF mutations suggesting that they are positively selected. Despite their prevalence, it is unclear whether these mutations have functional roles in cancer progression or are simply passenger mutations as the study of mtDNA mutations is stymied by the lack of methods to genetically modify the mtDNA. Here, I aim to test the hypothesis that heteroplasmic mtDNA mutations have functionally critical roles in tumorigenesis. In our preliminary work, we conducted single-cell RNAseq (scRNAseq) on Cal62, an anaplastic thyroid cancer cell line with three mtDNA loss-of-function (LOF) mutations. Using these data, I performed differential expression analysis between cells with high (0.8-1) and low (0-0.2) heteroplasmy levels which revealed an Epithelial-Mesenchymal-Transition (EMT) gene signature enriched in highly heteroplasmic cells. This raises the possibility that mtDNA mutations may result in a more invasive phenotype by impacting gene expression. In this proposal, I will elucidate the association between heteroplasmy and the EMT by creating an isogenic mtDNA mutant Cal62 cells and determine the impact of mtDNA mutations on cellular metabolism and growth. In this proposal, I will build upon our preliminary work to determine the association between mtDNA mutations and the EMT. In Aim1, I will identify transcripts that stratify cells of different heteroplasmy levels using scRNAseq in order to create isogenic mtDNA mutant cell lines. In Aim2, I will use the isogenic cell lines to determine the impact of mtDNA mutations on cellular metabolism and growth. I anticipate that these studies will determine: 1) the impact of mtDNA mutations on the cellular transcriptome and 2) the association of these mutations to cellular invasiveness. This work will be completed in the laboratory of Dr. Kvan Birsoy with the co-advisement of Dr. Andrew Clark at Rockefeller and Cornell University, respectively. The training plan outlined in this proposal is designed to best prepare me for a career as an independent scientist following my post-doctoral training.